Repositioning existing drugs for new therapeutic uses is an efficient approach to drugdiscovery. However, most successful repositioning cases to date have been serendipitous; thegoal of my thesis was to use computational methods to rationally discover drug repositioningcandidates.I first virtually screened (VS) 4621 drugs against 252 drug targets with molecular docking.This method emphasized removing potential false positives using stringent criteria fromknown interaction docking, consensus scores, and rank information. Published literatureindicated experimental evidence for 31 top predicted interactions, supporting the approach.The chemotherapeutic nilotinib was validated as a potent MAPK14 inhibitor in vitro (IC5040nM), suggesting a potential use in inflammatory diseases.I then applied this method to the cancer target EGFR, predicting the anti-HIV drug tenofovirdisoproxil fumarate (TDF) as a novel inhibitor. In vitro, TDF inhibited the proliferation andEGFR-signaling of an EGFR-overexpressing cell line, but did not inhibit EGFR in directkinase binding assays. This study highlighted limitations of computational and experimentalmethodologies that should be considered when interpreting or designing other studies.We then screened 1,120 off-patent drugs against the triple-negative breast cancer (TNBC)target p90RSK using both VS and high-throughput (HTS) methods. VS predicted a set ofcompounds 26-times enriched for known RSK inhibitors and 11 times enriched for HTS hits,underscoring its efficiency. In secondary screens, the chemotherapeutic ellipticine and thebioflavonoids luteolin and apigenin inhibited RSK activity (IC50 0.50-4.77μM), blocked RSKsignaling, and inhibited TNBC cell proliferation. These drugs thus have potential to berepositioned to TNBC.Finally, we rationally repositioned renal cell carcinoma drugs for a patient with a rare tongueadenocarcinoma. Whole genome and transcriptome sequencing of the patient’s tumor andnormal cells detected sequence, copy number, and expression aberrations, and analysis suggested that the tumor was driven by the RET oncogene. Treatment with RET-inhibitingdrugs stabilized the disease for eight months, after which the disease progressed. We alsosequenced the post-treatment tumor and found changes consistent with acquired therapeuticresistance.Overall, this thesis details two novel high-throughput approaches for drug repositioning:virtual screening of drugs and targets and personalized medicine via sequencing.